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[Cancer Research 66, 8749-8761, September 1, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Agonist and Antagonist of Retinoic Acid Receptors Cause Similar Changes in Gene Expression and Induce Senescence-like Growth Arrest in MCF-7 Breast Carcinoma Cells

Yuhong Chen1, Milos Dokmanovic1, Wilfred D. Stein1,2, Robert J. Ardecky3 and Igor B. Roninson1

1 Cancer Center, Ordway Research Institute, Albany, New York; 2 Institute of Life Sciences, Hebrew University, Jerusalem, Israel; and 3 Ligand Pharmaceuticals, Inc., San Diego, California

Requests for reprints: Igor B. Roninson, Cancer Center, Ordway Research Institute, 150 New Scotland Avenue, Albany, NY 12208. Phone: 518-641-6471; Fax: 518-641-6305; E-mail: Roninson{at}ordwayresearch.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Biological effects of retinoids are mediated via retinoic acid (RA) receptors (RAR) and retinoid X receptors (RXR). The best-characterized mechanism of retinoid action is stimulation of transcription from promoters containing RA response elements (RARE). Retinoids induce senescence-like growth arrest in MCF-7 breast carcinoma cells; this effect is associated with the induction of several growth-inhibitory genes. We have now found that these genes are induced by RAR-specific but not by RXR-specific ligands. Genome-scale microarray analysis of gene expression was used to compare the effects of two pan-RAR ligands, one of which is a strong agonist of RARE-dependent transcription, whereas the other induces such transcription only weakly and antagonizes the inducing effect of RAR agonists. Both RAR ligands, however, produced very similar effects on gene expression in MCF-7 cells, suggesting that RARE-dependent transcription is only a minor component of retinoid-induced changes in gene expression. The effects of RAR ligands on gene expression parallel changes associated with damage-induced senescence, and both ligands induced G1 arrest and the senescent phenotype in MCF-7 cells. The RAR ligands up-regulated many tumor-suppressive genes and down-regulated multiple genes with oncogenic activities. Genes that are strongly induced by RAR ligands encode secreted bioactive proteins, including several tumor-suppressing factors. In agreement with these observations, retinoid-treated MCF-7 cells inhibited the growth of retinoid-insensitive MDA-MB-231 breast carcinoma cells in coculture. These results indicate that RARE-independent transcriptional effects of RAR ligands lead to senescence-like growth arrest and paracrine growth-inhibitory activity in MCF-7 breast carcinoma cells. (Cancer Res 2006; 66(17): 8749-61)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Retinoids, natural and synthetic derivatives of vitamin A, regulate growth, differentiation, and survival of different types of normal and tumor cells. Retinoids are used in the treatment of promyelocytic leukemia and in chemoprevention of several cancers, including breast carcinoma. The antitumor effect of retinoids is most often attributed to the induction of differentiation, but these compounds were also shown to stop the growth of tumor cells by inducing apoptosis or accelerated senescence (1, 2). In particular, treatment of two human breast carcinoma cell lines with all-trans retinoic acid (RA) or fenretinide, in vitro or in vivo, induces a senescence-like phenotype characterized by increased cell size and expression of senescence-associated ß-galactosidase (SA-ß-gal; refs. 3, 4). This phenotype, as investigated in MCF-7 cells, is associated with irreversible growth arrest and up-regulation of several intracellular and secreted proteins with known growth-inhibitory activities. These include intracellular growth-inhibitory proteins, such as UBD (also known as FAT10) and putative tumor suppressor EPLIN, as well as secreted growth-inhibitory factors, including insulin-like growth factor-binding protein 3 (IGFBP3) and an extracellular matrix component, TGFBI also known as ßIG-h3 (ref. 4).

Induction of gene expression by retinoids is mediated at the level of transcription through binding to dimeric transcription factors formed by RA receptors (RAR) and retinoid X receptors (RXR). The best-known mechanism of action of these receptors involves their binding to RA response elements (RARE) in the promoters of retinoid-responsive genes. Nevertheless, retinoid receptors also affect transcription through RARE-independent mechanisms, such as repression of transcription factor activator protein (AP-1; Jun/Fos; ref. 5), or by modulating the interaction of Sp1 and GC-rich DNA via ternary complex formation (6). Remarkably, a survey of Balmer and Blomhoff (7) concluded that only a minority of all the published retinoid-inducible genes are induced through the RARE-dependent mechanism. In the case of retinoid-treated MCF-7 cells, only 1 of 13 genes found to be strongly up-regulated at the onset of senescence-like growth arrest contained a putative RARE sequence in its promoter, whereas the other genes had no identifiable RARE sites and showed a slow kinetics of retinoid response, requiring up to 3 days for maximal induction (4). Such genes may be induced either by an entirely RARE-independent mechanism or as a secondary consequence of some early RARE-dependent changes in gene expression.

In the present study, we have used pan-RAR– and pan-RXR–specific agonists and antagonists to investigate the roles of retinoid receptors in the induction of growth-inhibitory genes in MCF-7 cells. We have found that these genes are induced by the agonist of RAR (but nor RXR) and, surprisingly, by an RAR ligand that was developed as an antagonist of RARE-dependent transcriptional activation. Microarray analysis of gene expression showed that the agonist and the antagonist of RARE-dependent transcription produced very similar effects on global gene expression in MCF-7 cells. In agreement with these effects, both RAR agonist and RAR antagonist induced senescence-like growth arrest in MCF-7 cells. We have also identified numerous genes with oncogenic or tumor-suppressive properties that are affected by RAR ligands and shown that the up-regulation of secreted tumor-suppressive factors in retinoid-treated MCF-7 cells is associated with paracrine growth-inhibitory effect on retinoid-insensitive breast carcinoma cells. These results indicate that retinoids inhibit MCF-7 cell growth primarily through RARE-independent effects on cellular gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cellular assays. MCF-7 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in DMEM with 10% fetal bovine serum. Cells were plated at 105 per P100 (for assays requiring up to 6 days of culture) or at 104 per P60 (for longer assays), in the presence of different concentrations of all-trans-RA (from Sigma, St. Louis, MO), pan-RAR agonist LGD1550, pan-RXR agonist LGD1268, pan-RAR antagonist LG100815 or pan-RXR antagonist LG101208 (Ligand Pharmaceuticals, Inc., San Diego, CA), or DMSO carrier. Following treatment, the number of attached cells was measured using Coulter counter, and staining for SA-ß-gal was carried out as described (8). For cell cycle analysis, cells were stained with propidium iodide by standard procedures. Cellular DNA content was determined by flow cytometry using BD LSRII fluorescence-activated cell sorting, and the percentages of cells in G1, S, or G2-M were determined using ModFit software.

For coculture assays, MDA-MB-231 cells that were modified to express green fluorescent protein (GFP) from lentiviral vector pLL3.7 (9) were plated either alone (at 2 x 105 per P100) or mixed with MCF-7 (at 105 cells from each cell line). In some assays, MCF-7 cells pretreated with 100 nmol/L RA for 8 days were collected by trypsinization before mixing with MDA-MB-231. After culture in the presence or in the absence of 100 nmol/L RA, total cell number was counted and the percentage of MDA-MB-231 GFP cells was determined by flow cytometry.

Gene expression analysis. MCF-7 cells were plated at 5 x 105 per P100; exposed to different drugs or carrier on the next day; and cells were collected after 24, 48, or 72 hours treatment. Total cellular RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA). Gene expression levels of IGFBP3, EPLIN, UBD, TGFBI, and TRIM31 were quantitated by reverse transcription-PCR (RT-PCR) as described (4) and by quantitative real-time RT-PCR (QPCR), using an ABI 7900HT real-time PCR instrument. In QPCR, serial cDNA dilutions were used for primer validation and the comparative CT method for relative quantitation of gene expression (Applied Biosystems, Foster City, CA) was used to determine expression levels for target genes. ß-Actin was used as a normalization standard. Primer sequences will be provided upon request. For gene expression profiling, RNA samples were provided to the Microarray Core Facility at the Genomics Institute of the NYSDOH Wadsworth Center, which carried out biotinylated target preparation (using 2 µg RNA per assay) and hybridization with Affymetrix U133 Plus 2.0 microarrays. Data analysis was carried out using GeneSpring software (Agilent, Palo Alto, CA). Gene function analysis was carried out using Pathway Assist (Ariadne Genomics, Rockville, MD) and PubMed.

RARE-dependent transcription was analyzed using a plasmid construct that expresses firefly luciferase from a RARE-containing artificial promoter DR5 (Stratagene, La Jolla, CA). Cells were plated at 3 x 105 per P60 24 hours before transient transfection. DR5 reporter plasmid (4 µg) was mixed with the SV40-driven Renilla luciferase control plasmid (0.04 µg) and transfected using LipofectAMINE Plus (Invitrogen, Carlsbad, CA) as described by the manufacturer. Three hours after transfection, cells were rinsed thrice with PBS, trypsinized, and replated in a 12-well plate to the density of 5 x 104 per well. Retinoid agonists or antagonists were added 48 hours later, and the luciferase assay was done after another 24 hours.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RAR agonists and an antagonist induce the expression of growth-inhibitory genes. To determine which classes of retinoid receptors mediate the induction of growth-inhibitory genes in retinoid-treated MCF-7 cells, we have analyzed the effects of pan-RAR– and pan-RXR–specific agonists and antagonists on these cells. The tested compounds include LGD1550, a pan-RAR agonist that shows greater selectivity for RAR than natural retinoids (10); LGD1268 (a pan-RXR agonist); LG100815 (a pan-RAR antagonist); and LG101208 (a pan-RXR antagonist). We have also used all-trans-RA, a natural RAR agonist that was originally used to define retinoid-induced senescence and changes in gene expression in MCF-7 cells (3, 4). The structures of these compounds are shown in Fig. 1 .


Figure 1
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  		Figure 1. Structures of retinoid receptor ligands used in the present study. Structure representations in two-dimensional and three-dimensional formats were generated using ChemDraw Pro version 10.0 and Chem3D Ultra version 10.0, respectively.

 
In the first set of experiments, we asked how these compounds affect the expression of several growth-inhibitory genes previously found to be strongly up-regulated under the conditions of retinoid-induced senescence of MCF-7 cells. The tested genes included EPLIN-ß, UBD (also known as FAT10), IGFBP3, and TGFBI (also known as ßIG-h3), as well as TRIM31, a gene strongly induced by RA that has a putative RARE element in its promoter (4). RNA was extracted from MCF-7 cells that were either untreated or treated with individual compounds or their combinations for 3 days, the period previously shown to be required for the maximal effect of RA or fenretinide (4). Gene expression was analyzed both by semiquantitative RT-PCR (not shown) and by QPCR; both assays produced essentially the same results. The outcome of a representative set of QPCR assays is shown in Table 1 .


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  		Table 1. QPCR analysis of the effects of retinoid receptor ligands on gene expression

 
In agreement with the previous study, all five of the tested genes were induced by 100 nmol/L RA, the concentration found to be sufficient for maximal induction of gene expression (4), with TRIM31 showing greater fold induction than the other four genes. The pan-RAR agonist LGD1550 induced all five genes as strongly as RA; a 100 nmol/L concentration of this agonist was also sufficient for the maximal effect. In contrast, the pan-RXR agonist LGD1268 showed detectable induction of only two of the five genes (TGFBI and TRIM31), and the extent of induction was much lower than observed for RA or the pan-RAR agonist. The pan-RXR antagonist LG101208 showed no effect on the expression of any of the five genes (Table 1). When combined with 100 nmol/L RA, 10 µmol/L of the pan-RXR antagonist inhibited the induction of only one of these genes (IGBP3) by RA (Table 1). These findings suggest that retinoid-inducible expression of growth-inhibitory genes is activated primarily through RAR rather than RXR.

Surprising results were obtained, however, with the pan-RAR antagonist LG100815. This compound acts as a competitive inhibitor of RAR agonists, which binds to RAR but does not efficiently activate RARE-dependent transcription (11).4 To verify the antagonistic effect of LG100815 on RARE-dependent transcription, we analyzed the effects of this compound and of RAR agonists RA and LGD1550 on the expression of firefly luciferase reporter transcribed from a RARE-containing artificial promoter, DR5. Figure 2 shows the results of DR5-luciferase transient transfection assays, carried out in the presence of LGD1550, RA, and LG100815, alone or in pairwise combinations. One hundred nanomoles per liter concentrations of RA or LGD1550 agonists activated the RARE-containing promoter ~50-fold, whereas 10 µmol/L LG100815 antagonist (the concentration used in the literature for maximal effect) produced an order of magnitude weaker (4.2-fold) induction. On the other hand, the addition of LG100815 to RAR agonists RA or LGD1550 diminished the induction of transcription by the latter compounds 2.5 to 3 times (Fig. 2). These results confirm that LG100815 is relatively inefficient in stimulating RARE-dependent transcription and that it antagonizes the effect of RAR agonists on such transcription.


Figure 2
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  		Figure 2. Effects of RAR agonists (RA and LGD1550, 100 nmol/L each) and RAR antagonist LG100815 (10 µmol/L) on luciferase expression from DR5 RARE-containing promoter in MCF-7 cells. The assays were carried out as described in Materials and Methods, in triplicate.

 
In contrast to the results of the promoter assays, QPCR analysis showed that LG100815 antagonist induced RNA expression of all the tested endogenous genes to a level comparable with RAR agonists (Table 1). The fold induction of these genes by 1 or 10 µmol/L LG100815 was only slightly lower than the highest induction by either RA or LGD1550 agonist, with the only large difference (3- to 4-fold) observed for TRIM31. When 10 µmol/L LG100815 was combined with RA, it decreased the induction of gene expression by RA to the levels that were similar to those observed with the antagonist alone, as expected for a competitive effect (Table 1). Hence, LG100815, although acting as a weak inducer and a competitive antagonist of RARE-dependent transcription, mimics the effects of RAR agonists in stimulating the expression of the tested growth-inhibitory genes. This surprising observation prompted us to compare the effects of the RAR agonist and the RAR antagonist on the expression of other genes and on the phenotype of MCF-7 cells. In most of the subsequent studies, we used 100 nmol/L LGD1550 agonist and 10 µmol/L LG100815 antagonist, the concentrations that produced the maximal effects in the above-described assays.

RAR agonist and antagonist produce similar effects on genome-scale gene expression. To analyze the effects of RAR ligands on the expression of essentially all the cellular genes, we treated MCF-7 cells with RAR agonist LGD1550 (100 nmol/L) or RAR antagonist LG100815 (10 µmol/L) for 24, 48, or 72 hours, and analyzed RNA from the untreated or treated cells by hybridization with Affymetrix U133 Plus 2.0 oligonucleotide arrays, containing 56,000 probe sets representing 48,500 human transcripts. The time course of changes in gene expression observed in the microarrays (Fig. 3A ) was in agreement with the results of RT-PCR assays (not shown) and with previous studies on RA- or fenretinide-treated MCF-7 cells (4), with most of the responsive genes showing the response on day 1 with subsequent increases up to day 3. All 13 genes shown by RT-PCR to be induced by RA or fenretinide (4) also showed induction by the pan-RAR agonist and antagonist (Fig. 3B).


Figure 3
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  		Figure 3. Microarray analysis of changes in gene expression in MCF-7 cells treated with RAR agonist LGD1550 or RAR antagonist LG100815, plotted using GeneSpring software. X axis, different time points of treatment with RAR ligands (0 point correspond to cells cultured for 3 days with DMSO carrier). Y axis, changes in gene expression on log scale. The groups of genes shown in (A), (G), and (H) represent GO categories, with the exclusion of genes showing raw signal intensity <10 in MCF-7 cells. The selection of the other gene groups is described in the text.

 
Strikingly, the effects of the RAR agonist and the RAR antagonist were exceedingly similar. Seventy-four percent of the genes showing ≥1.5-fold induction and 77% of the genes showing ≥1.5-fold inhibition by the antagonist were also induced or inhibited, respectively, at least 1.3-fold by the agonist, and vice versa (77% and 69%, respectively). Figure 4A plots (on the log scale) the maximal changes in gene expression (at any time point) produced by the agonist versus those produced by the antagonist for 11,729 probe sets that showed >1.3-fold changes in gene expression after treatment with either the agonist or the antagonist. The effects of the agonist and antagonist on gene expression show highly significant correlation. The regression through all the data points has an R2 value of 0.6955 with n = 11,729 (Student's t test yields P < 0.0001). The regression line has a slope of 1.027 ± 0.006, indicating that the RAR agonist and the RAR antagonist have the same effect on the majority of the responsive genes. The similarity of the overall effects of the agonist and the antagonist on gene expression agrees with the results of QPCR analysis of selected genes (Table 1), but contrasts with an order of magnitude weaker effect of the antagonist on RARE-dependent transcription (Fig. 1).


Figure 4
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  		Figure 4. Comparison of changes in gene expression produced by RAR agonist and antagonist. The maximal changes in gene expression for 11,729 probe sets representing genes that show >1.3-fold (A) or >5-fold (B) effect by either the agonist or the antagonist (dots) are plotted on a log scale. Trend lines represent power regression. Gene names for selected probe sets strongly affected by either ligand are shown in (B).

 
On the other hand, 316 probe sets showing the strongest (>5-fold) induction or inhibition by RAR ligands were significantly more responsive to the agonist than to the antagonist (at P < 0.0001), with the regression slope increasing to 1.297 ± 0.038 (this translates to ~2-fold stronger average effect of the agonist relative to the antagonist; Fig. 4B). We considered whether preferential induction of the most responsive genes by the agonist could indicate the presence of RARE sequences in the corresponding promoters. Indeed, the gene showing the strongest induction by the agonist (Fig. 4B) encodes RA-metabolizing enzyme CYP26A1 (induced 220-fold by the agonist and 23.5-fold by the antagonist), which was reported to contain two synergistically acting RARE sequences in its promoter (12). Another cytochrome P450 involved in RA metabolism, CYP26B1, showed the most selective response to the RAR agonist among all the genes (180-fold induction by the agonist, compared with only 2.4-fold induction by the antagonist). We have also looked at the effects of the agonist and the antagonist on the expression of 40 human genes, identified by Balmer and Blomhoff (13) as containing canonical and evolutionarily conserved RARE sequences in their promoters. Only 7 of these 40 genes were induced ≥1.5-fold in MCF-7 cells by the RAR agonist and just three genes were induced by the antagonist. RARE-containing genes induced by both ligands showed 3- to 5-fold stronger response to the agonist than to the antagonist (Fig. 3C). The most responsive gene in this group is HOXA1, induced 95-fold by the agonist but only 18-fold by the antagonist. However, when we examined promoter sequences of 10 randomly chosen genes that show preferential induction by the agonist for the presence of putative RARE sequences (this analysis was carried out using MatInspector program as previously described; ref. 4), only 1 of 10 promoters was found to contain putative RARE sequences. We also examined 28 randomly selected genes from the group identified by Balmer and Blomhoff (7) as indirectly inducible by retinoids ("category 0") and found that these genes showed a similar response to the agonist and the antagonist (Fig. 3D and data not shown). Altogether, these results indicate that the majority of genes induced by the RAR ligands are induced through indirect, RARE-independent mechanisms.

RAR agonist and antagonist induce senescence-associated changes in gene expression and cellular phenotype. MCF-7 cells treated with 100 nmol/L RA eventually (after ≥4 days) undergo cytostatic growth arrest, which is accompanied by the loss of clonogenic potential upon removal of the drug and the development of the senescent phenotype (3, 4). We compared changes in gene expression induced in MCF-7 cells by LGD1550 and LG100815 with the results obtained in a well-characterized system of drug-induced senescence of tumor cells. In that system, HCT116 colon carcinoma cells were transiently exposed to doxorubicin, the surviving cells were separated into proliferating and senescent populations after drug treatment, and differentially expressed genes were identified using a cDNA microarray (14). We have recently repeated this analysis using Affymetrix U133 Plus 2.0 array.5 We now asked how treatment of MCF-7 cells with RAR ligands affects the expression of genes found in the latter analysis to be strongly (>5-fold) induced or inhibited in senescent HCT116 cells. As shown in Fig. 3E and F, most of these genes changed their expression in the same direction in MCF-7 cells treated with RAR agonist or antagonist. Specifically, 88% of 231 genes down-regulated in senescent HCT116 cells and expressed in MCF-7 cells were down-regulated by the RAR agonist, and only 3% were up-regulated. Among 353 genes up-regulated in senescent HCT116 cells and expressed in MCF-7, 53% were up-regulated by the agonist and 10% were down-regulated. Similar results were obtained with the RAR antagonist. This analysis indicates profound similarities between the effects of retinoids and doxorubicin-induced senescence on gene expression. On the other hand, there were also notable differences in gene expression between RAR ligand-treated MCF-7 cells and HCT116 cells undergoing doxorubicin-induced senescence. These differences may be attributed to a large extent to the fact that only the latter but not the former up-regulated cyclin-dependent kinase (CDK) inhibitor p21Waf1 (CDKN1A), a key regulator of gene expression in senescent cells (see Discussion).

The results of gene expression studies suggested that RAR agonist and antagonist (but not RXR ligands) are likely to induce senescence-like growth arrest in MCF-7 cells. To test this, we treated MCF-7 cells with RAR and RXR agonists and antagonists for 7 days and analyzed the effects of the treatment on the cell number (Fig. 5A ), cell cycle distribution (Fig. 5B), and the expression of the SA-ß-gal marker of senescence (Fig. 5C, D; ref. 8). The RXR agonist LGD1268 did not inhibit MCF-7 cell growth (Fig. 5A) and did not induce the senescent phenotype (Fig. 5D); in fact, LGD1268 treatment produced a moderate but reproducible increase in cell growth (Fig. 5A). The RXR antagonist LG101208 had no effect on either the cell growth (Fig. 5A) or the senescent phenotype (Fig. 5D). In contrast, the RAR agonist LGD1550 and the RAR antagonist LG100815 inhibited cell growth to the extent similar to that of RA (Fig. 5A). Cell cycle analysis showed that RA, LGD1550, and LG100815 all increased the G1 and decreased the S fraction, indicating cell cycle arrest in G1 (Fig. 5B), in agreement with previous observations on RA-treated MCF-7 cells (15). LGD1550 and LG100815 increased SA-ß-gal activity (Fig. 5C), as has also been shown for RA (3), and this effect of all three RAR ligands was quantitatively similar (Fig. 5D). Hence, RAR agonists and the antagonist induce senescence-like growth arrest in MCF-7 cells with a similar efficiency.


Figure 5
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  		Figure 5. Effects of retinoid agonists and antagonists on MCF-7 cell growth and the senescent phenotype. A, cell number after 7 days of culture with the addition of DMSO (control), 100 nmol/L RA, 100 nmol/L RAR agonist LGD1550, 100 nmol/L RXR agonist LGD1268, 10 µmol/L RAR antagonist LG100815, and 10 µmol/L RXR antagonist LG101208. Experiments were done in triplicate, and the results are expressed relative to the average of the control. B, changes in cell cycle distribution in untreated cells or in cells treated with 100 nmol/L RA, 100 nmol/L LGD1550 RAR agonist, 10 µmol/L LG100815 RAR antagonist, or DMSO carrier (untreated). For cells treated with RAR ligands, 0 point represents cells cultured for 2 days with DMSO carrier. The analysis was carried out as described in Materials and Methods. C, staining of cells that were treated for 8 days with DMSO carrier (control), 100 nmol/L LGD1550, or 10 µmol/L LG100815 for senescence marker SA-ß-gal. Photographed at x100 magnification. D, percentages of SA-ß-gal+ cells after 8 days of treatment with the indicated compounds (in triplicate), at the same concentrations as in (A).

 
Effects of RAR ligands on tumor suppressor and oncogene expression and paracrine growth-inhibitory activity of retinoid-treated MCF-7 cells. The most prominent Gene Ontology categories of genes that were largely inhibited by RAR ligands are genes involved in DNA replication (Fig. 3G) or mitosis (Fig. 3H). Strong down-regulation of such genes has been associated with cell cycle arrest induced by chemotherapeutic drugs. In contrast, inhibition of these genes by RAR ligands was moderate (~2-fold), with none of the genes showing ≥5-fold inhibition. On the other hand, we noticed that many of the genes that show the greatest fold inhibition by RAR ligands are known or putative oncogenes. (Here, we define genes as putative oncogenes or tumor suppressors if such genes have been reported in the literature to play a functional role in tumor cell growth, survival, tumorigenesis, or metastasis, as determined by targeted inhibition, gene overexpression or protein addition studies.) A total of 26 oncogenes were found to be inhibited by RAR ligands, with the strongest inhibition found for VAV3, SPDEF (also known as PDEF), AMIGO2, MYB, RET, C4.4A, and MAFB (Table 2 ). On the other hand, we also identified a smaller number (10) of genes with reported tumor-suppressive activities that were inhibited by RAR ligands (Table 2), most notably caveolin proteins CAV1 and CAV2, as well as cellular RA-binding protein 2 (CRABP2), a retinoid-binding protein reported to sensitize MCF-7 cells to growth inhibition by RA (16). We have also identified 22 known or putative oncogenes and 34 tumor suppressors as up-regulated by RAR ligands (Table 3 ). The products of these genes include both secreted factors (see below) and cell-associated proteins. The most highly induced cell-associated growth inhibitors were the previously identified retinoid-inducible genes UBD, EPLIN (4), and SOX9 (17), followed by CEACAM1, PPARG, MARCKS, BTG2, and NKX3-1. The most strongly induced oncogenes were the RARE-regulated transcription factor HOXA1 and RGC32, a positive regulator of the cell cycle. The time course of the induction or inhibition of cell-associated oncogenes and tumor suppressors listed in Table 2 is shown in Fig. 3I and J, respectively.


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  		Table 2. Oncogenes and tumor suppressors inhibited by RAR ligands

 

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  		Table 3. Oncogenes and tumor suppressors induced by RAR ligands

 
Many genes up-regulated by RAR ligands encode secreted proteins. Some of these proteins have cancer-relevant activities, stimulating or inhibiting cell growth, survival, invasion, or angiogenesis. The induced secreted proteins with tumor-promoting (oncogenic) or tumor-suppressing activities are listed in Table 3, whereas the inhibited secreted proteins are listed in Table 2. Figure 3K and L show the effects of RAR ligands on the expression of the corresponding genes. The genes for secreted proteins showing the strongest induction include four tumor-suppressing factors (TGFBI, IGFBP3, FBLN5, and GDF15) and two tumor-promoting proteins [interleukin-8 (IL-8) and PLAG2A].

Because retinoid-treated MCF-7 cells up-regulate genes for secreted factors with different activities, we carried out a functional test to determine whether such cells produce primarily promitogenic or antimitogenic paracrine effects. In this assay, we mixed MCF-7 cells 1:1 with MDA-MB-231 breast carcinoma cells (insensitive to retinoids). The latter cells had been transduced with GFP, allowing us to distinguish and quantitate the two cell lines by flow cytometry. The cocultures were treated for 5 days with 100 nmol/L RA (used because of limited availability of LGD1550 and LG100815). RA or MCF-7 cells alone had no effect on the growth of MDA-MB-231 cells. In contrast, retinoid treatment in coculture with MCF-7 decreased the number of MDA-MB-231 cells by ~25% (Fig. 6A ). In another type of experiment, shown in Fig. 6B, MCF-7 cells were pretreated for 8 days with RA to allow for complete growth arrest and development of the senescent phenotype. The treated MCF-7 cells were collected by trypsinization and cocultured for 3 days with MDA-MB-231, in the presence or in the absence of RA. Coculture with RA-pretreated MCF-7 cells was sufficient to inhibit MDA-MB-231 cell growth by ~30%, compared with coculture with untreated MCF-7 cells (Fig. 6B). The addition of RA to the coculture had no significant effect on this growth inhibition (Fig. 6B), indicating that MDA-MB-231 cell growth was inhibited not by the retinoid but by factors secreted by retinoid-treated MCF-7 cells.


Figure 6
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  		Figure 6. Effects of coculture with RA-treated MCF-7 cells on MDA-MB-231 cell growth. A, MDA-MB-231 cells (GFP-expressing) were plated either alone or in 1:1 mixture with MCF-7 cells, in the presence of 100 nmol/L RA or DMSO carrier. Columns, mean MDA-MB-231 cell number after 5 days of culture relative to cell number in the absence of MCF-7 or RA, calculated from three independent experiments; bars, SD. B, MDA-MB-231 cells (GFP-expressing) were plated as 1:1 mixtures with MCF-7 cells that were either untreated or treated for 8 days with 100 nmol/L, and grown in the presence of DMSO carrier or 100 nmol/L RA. Columns, mean MDA-MB-231 cell number relative to MDA-MB-231 cell number in coculture with untreated MCF-7 without RA, calculated from three independent experiments; bars, SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have used pan-RAR– or pan-RXR–specific agonists and antagonists to investigate the mechanism of changes in gene expression associated with senescence-like growth arrest, which is induced by retinoids in MCF-7 breast carcinoma cells. Biological effects of retinoids are commonly attributed to the effects of ligand-bound retinoid receptors on transcription from RARE-containing promoters. However, the majority of genes induced by retinoids in different systems (7), including MCF-7 cells treated with RA or fenretinide (4), do not contain RARE elements in their promoters. Nevertheless, RARE-mediated induction of transcription could still be the primary response to retinoids, triggering a chain of events leading to indirect changes in the expression of other genes. The results of the present study suggest, however, that RARE-dependent transcription plays only a minor role in the effects of retinoids on gene expression in MCF-7 cells, and that changes in gene expression responsible for senescence-like growth arrest are due primarily to RARE-independent transcriptional effects.

These conclusions are based on the following arguments. (a) The limited role of RARE promoter sequences in determining transcriptional effects of retinoids is indicated by the findings that only a small fraction of the genes induced by RAR ligands contain RARE elements in their promoter, and that the majority of RARE-containing genes examined are not affected by the RAR ligands. (b) RARE-dependent transcription is also unlikely to be the initial effect responsible for subsequent global changes in gene expression, as indicated by the comparison of the effects of RAR agonist LGD1550 and RAR antagonist LG100815. The RAR agonist was an order of magnitude more efficient than the antagonist in stimulating a RARE-containing promoter (at concentrations producing the maximal effects), and it induced the responsive genes known to contain functional RARE sequences 3 to 10 times stronger than the antagonist. One would expect therefore that genes that are induced as a consequence of RARE-dependent early events should also show preferential response to the agonist, even if such genes do not contain RARE elements. Indeed, we have identified a set of genes, primarily among the strongest responders, which are preferentially induced or inhibited by the agonist relative to the antagonist. Such genes, however, were a small minority (e.g., 1,616 genes were up-regulated >2-fold by either ligand, but only 261 of these genes showed ≥2-fold stronger response to the agonist than to the antagonist), and the average effects of the agonist and the antagonist on the bulk of the responsive genes were essentially the same (Fig. 4A). (c) Nevertheless, one could still argue that RARE-dependent transcription, which is preferentially induced by the agonist, could be the key determinant of the effect of retinoids on the growth of MCF-7 cells, whereas RARE-independent changes in global gene expression could be epiphenomena irrelevant to the biological response. However, the RAR antagonist LG100815 was just as efficient as the RAR agonists (RA and LGD1550) in inducing cell growth inhibition, G1 arrest, and the senescent phenotype (Fig. 5).

A number of RARE-independent mechanisms of regulation of gene expression by retinoids have been described in the literature. Not all of these mechanisms are transcriptional; for example, retinoids were suggested to affect RNA stability (18). We have analyzed mRNA stability of IGFBP3, TGFBI, UBD, and EPLIN during 24-hour treatment with actinomycin D and found these messages to be as stable as ß-actin mRNA, with no detectable effect of RA on their stability (19). Hence, the effect of retinoids on these genes is more likely to be exerted at the level of transcription. In the case of IGFBP3, the induction of transcription by RA in MCF-7 cells has been shown by nuclear run-on assays (20). Some examples of RARE-independent transcription regulatory mechanisms include the binding of RAR/RXR–based transcription factor complexes to cis-acting sequences distinct from RARE (21) or interactions between retinoid receptors and other transcription factors, such as Sp1 (6, 22) or AP-1 (5, 23). Furthermore, retinoids can regulate the activity of protein kinase C (PKC; ref. 24), and RA-activated PKC was shown to bind to RAR and stimulate its transcriptional activity (25). The effects of RAR ligands on gene expression, observed in the present study, most likely reflect many different RARE-independent transcriptional effects of retinoids rather than any single mechanism.

In light of our findings, it would seem more appropriate to describe compounds such as LG100815 not as RAR antagonists but rather as a novel type of RAR modulators that are poor inducers of RARE-dependent transcription and that act primarily by stimulating RARE-independent transcriptional effects. Not all the RAR antagonists seem to belong to this class. For example, a RAR-{alpha}–specific antagonist LG100629 was shown to block essentially completely the induction of IGFBP3 and TGFB2 by RA in human bronchial epithelial cells (26), in contrast to our findings with pan-RAR modulator LG100815. On the other hand, several RAR antagonists, similarly to LG100815, were found to inhibit tumor cell growth (27–30). It would be of interest to determine if these compounds act as LG100815-type RAR modulators.

Genome-scale microarray analysis of the effects of RAR ligands has confirmed and expanded the results of our previous analysis of the effects of RA in MCF-7 cells, carried out using a smaller microarray (4). The principal conclusion of the prior study was that retinoid-induced arrest of MCF-7 cells was associated with concerted induction of several growth-inhibitory genes encoding both cell-associated (UBD and EPLIN) and secreted proteins (TGFBI and IGFBP3). The present analysis showed that RAR ligands LGD1550 and LG100815 also strongly induced these genes, and it revealed many additional tumor-suppressive genes induced by retinoids (e.g., SOX9, CEACAM1, MARCKS, GDF15, FBLN5, BTG2, NKX3-1, NBL1, and IRF1). Importantly, we discovered in the present study that RAR ligands not only induce tumor suppressors but also inhibit multiple oncogenes or proto-oncogenes expressed in MCF-7 cells, with the strongest effects observed for VAV3, SPDEF, AMIGO2, MYB, RET, and C4.4A. We have investigated the effects of overexpressing several retinoid-inducible growth-inhibitory genes, including UBD (4), EPLIN, and TGFBI, as well as IGFBP3 protein product (19), on MCF-7 cells, and found that each of these genes produced only weak growth inhibition and did not induce the senescent phenotype. In light of these findings, we hypothesize that retinoids induce senescence-like growth arrest in MCF-7 cells through a cumulative effect of multiple changes in the expression of different growth-regulatory genes.

Many of the genes found in the present study to be affected to a similar extent by RAR agonist and antagonist (i.e., RARE-independent genes) have been previously reported to be retinoid responsive in MCF-7 and other breast carcinoma cell lines. Of special interest, these include tumor suppressor genes proposed to mediate the antiproliferative effects of retinoids, such as IGFBP3, induced by RA in MCF-7 and Hs578T lines (31), as well as SOX9 and PDCD4, induced by RAR-specific but not by RXR-specific agonists in MCF-7 and T-47D cells (17, 32). Other genes identified by our microarray analysis have been implicated specifically in the phenotype of breast carcinoma cells, including MCF-7. Among the genes that we found to be inhibited by retinoids, ets transcription factor SPDEF (PDEF) was shown to be a key mediator of motility and invasion in MCF-7 and other breast carcinoma cell lines (33), PDLIM2 (Mystique) is a negative regulator of anchorage-independent growth and migration in MCF-7 cells (34), and GREB1 was reported to mediate the proliferative response of MCF-7 cells to estrogen (35). Among the genes found here to be induced by RAR modulators, an isoform of adhesion molecule CEACAM1 inhibits cell growth and restores normal morphology of MCF-7 cells (36), and IRF1 was shown to mediate p53-independent tumor suppression and apoptosis in MCF-7 and other breast carcinoma cell lines (37). Growth inhibition and apoptosis in retinoid-treated MCF-7 cells have been previously associated with the drastically overexpressed IGFBP3 (38) and with the retinoid-binding protein CRABP2 (39), which, paradoxically, we find here to be strongly down-regulated rather than induced by RAR ligands. Down-regulation of CRABP2 gene expression upon retinoid treatment could potentially represent a negative regulatory mechanism that limits retinoid-induced apoptosis. Another surprising observation is the inhibition of caveolin proteins CAV1 and CAV2. Caveolins were shown to act as tumor suppressors in the mammary gland but as oncogenes in other tissue contexts, such as the prostate (40). CAV1 expression in MCF-7 cells was found to inhibit anchorage-independent growth but at the same time protect cells from apoptosis (41). Altogether, despite some changes to the contrary, the overall effect of RAR modulators on the expression of oncogenes and tumor suppressors in MCF-7 cells seems to be indicative of the reversal of the neoplastic phenotype.

Several retinoid-induced tumor suppressors (BTG2, BTG1, GDF15, EPLIN, and CEACAM1) are also induced by DNA-damaging drugs (such as doxorubicin) and remain constitutively up-regulated in HCT116 colon carcinoma cells that become permanently growth arrested and develop the senescent phenotype after exposure to doxorubicin (14). Aside from these genes, we have found a striking overlap between the large groups of genes that are induced or inhibited in doxorubicin-induced senescence of HCT116 cells and in retinoid-induced senescence of MCF-7 cells (Fig. 3K and L). On the other hand, an important difference between retinoid-treated MCF-7 cells and most of the characterized systems of DNA damage–induced senescence is the lack of induction of the damage-responsive CDK inhibitor p21Waf1 (CDKN1A), which was found here and in previous studies (15) to be moderately down-regulated in MCF-7 cells after retinoid treatment. In fact, the only CDK inhibitor that we found to be up-regulated in retinoid-treated MCF-7 cells was p15Ink4b (CDKN2B), which was recently identified as a marker of oncogene-induced senescence (42). In contrast, p21 is drastically induced in MCF-7 cells after doxorubicin treatment, concordantly with the development of the senescent phenotype (43). p21 induction leads to rapid inhibition of genes involved in mitosis or DNA replication (44), and p21 knockout prevents the shutdown of such genes in doxorubicin-treated cells (14). Although genes involved in mitosis and DNA replication are clearly inhibited in RAR ligand–treated MCF-7 cells (Fig. 2F and G), their inhibition is not as drastic as in DNA-damaged cells (14), possibly due to the low levels of p21. The lack of p21 induction is also likely to account for our observations that a number of genes that are induced in response to p21 and implicated in tumor promotion or the development of age-related diseases, such as LGALS3, APP, or SHC1 (44), were not induced in RAR modulator–treated MCF-7 cells.

Induction of senescence-like permanent growth arrest could be one of the most desirable treatment responses in tumor cells, because it occurs at low drug concentrations that produce little systemic toxicity, and because senescent cells not only fail to grow but also secrete proteins with paracrine growth-inhibitory effects. On the other hand, senescence induced by treatment with p21-inducing DNA-damaging agents is also associated with overproduction of proteins with the opposite, tumor-promoting activities, which stimulate the growth or survival of the neighboring nonsenescent cells (45). In our previous study on retinoid-induced senescence of MCF-7 cells (4), we observed overproduction of secreted growth-inhibitory proteins (TGFBI and IGFBP3). We have now found that MCF-7 cells treated with RAR ligands up-regulate not only additional tumor-suppressing proteins (GDF15 and FBLN5) but also tumor-promoting factors (IL-8 and PLAG2A), although tumor-suppressing proteins show overall greater induction, at least at the RNA level (Fig. 3K and L). The results of our coculture studies with retinoid-sensitive and retinoid-insensitive breast carcinoma cell lines (Fig. 6) show that retinoid-treated MCF-7 cells indeed secrete tumor-suppressing factors that produce a moderate but reproducible decrease in the growth of neighboring retinoid-insensitive MDA-MB-231 cells. Exploitation of this paracrine tumor-suppressive effect of retinoid-treated tumor cells offers a new insight into the clinical applications of retinoids.

Despite the shown efficacy of retinoids in PML treatment and in cancer chemoprevention trials, retinoid treatment is complicated by systemic toxic responses, such as intracranial hypertension and headaches, dyspnea, hypertriglyceridemia, hypercalcemia, and hyperleukocytosis (46). An experimental approach proposed to avoid these side effects is the use of RXR-selective agonists that show lower toxicity than the conventional RAR agonists (47). However, RXR agonists may not be effective against all the types of transformed mammary epithelial cells, as indicated in the present study by the observation that LGD1268 did not inhibit but rather stimulated MCF-7 cell growth (Fig. 5A). As an alternative approach, RAR antagonists have been developed to prevent or treat retinoid toxicity (48). RAR antagonists were reported to be very well tolerated even at very high doses (49) and in some cases produced in vivo tumor-suppressive effects on their own (49, 50). The results of the present study suggest an explanation for these observations. LG100815-type RAR modulators are inefficient in inducing the major effect of conventional retinoids (RARE-dependent transactivation), which provides the likeliest explanation for their nontoxicity. On the other hand, LG100815 mimics the effects of conventional RAR agonists in inducing senescence-like growth arrest of tumor cells and up-regulating the expression of genes for tumor-suppressing secreted factors. This novel type of RAR modulators may be viewed therefore as a prototype of a potentially interesting new class of drugs that should provide a high therapeutic ratio for cancer treatment or chemoprevention.


   Acknowledgments
 
Grant support: NIH grants R01 CA62099 and R01 AG17921 (I.B. Roninson).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. William Lamph (Ligand Pharmaceuticals, Inc., San Diego, CA), for providing RAR and RXR ligands used in this study and for helpful advice; Dr. Chang Lim for assistance with flow cytometry; Drs. Eugenia Broude, Errin Lagow, and Charitha Madiraju for helpful discussions; and the late Dr. Robin Pietropaolo and the Microarray Core Facility at the Genomics Institute of the New York State Department of Health Wadsworth Center, who carried out microarray hybridization.


   Footnotes
 
Note: Current address for Milos Dokmanovic: Memorial Sloan-Kettering Cancer Center, Cell Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY 10021.

4 W. Lamph, personal communication. Back

5 Y. Chen and I.B. Roninson, in preparation. Back

Received 2/14/06. Revised 5/22/06. Accepted 6/28/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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